The goal of this project is to understand the roles of RNA binding proteins in the post-transcriptional regulation of gene expression, through an analysis of the in vivo functions of proteins that contain RNA recognition motif (RRM) domains. RRM domains have been identified in many proteins involved in post-transcriptional regulation, and can mediate sequence-specific binding to RNA targets. We use genetics, biochemistry and molecular biology to study six Drosophila RRM proteins. Two of these, TSR and RB97D, are expressed during spermatogenesis, and are required for normal male fertility. The Tsr gene is expressed only in the male germ line, and mutations in Tsr drastically reduce male fertility. Electron microscopic analysis of testes from Tsr mutants reveals dramatic defects during spermatid morphogenesis. The mitochondria develop abnormally, with an aberrant relationship to the axoneme of the sperm tail. TSR is a cytoplasmic protein and is present at high levels in older primary spermatocytes and young spermatids. Translational regulation is known to be critical during spermatogenesis, and TSR may be involved in this process. We have used the yeast two- hybrid system to identify other proteins that may interact with TSR. One protein that specifically interacts with TSR is the ADP-ribosylation factor (ARF), a small GTP binding protein. ARF proteins have been implicated in membrane trafficking and organelle integrity, and a role for ARF in the remodeling of the spermatid mitochondrion is plausible. The TSR-ARF interaction might serve to integrate mitochondrial morphogenesis with the post-transcriptional regulation of specific mRNAs. Mutations in the Rb97D gene cause male sterility. Rb97D encodes a nuclear protein that binds to the ks-1 lampbrush loop of the Y chromosome in primary spermatocytes. The ks-1 loop is a transcriptionally active region containing a gene essential for male fertility. The binding of RB97D is RNAse-sensitive, suggesting association with ks-1 transcripts. When transcription ceases and the loop regresses late in spermatogenesis, RB97D is no longer detectable. These data are consistent with the idea that RB97D is required for processing nascent transcripts from the ks-1 gene.